DZL scientists, together with working groups from the Universities of Giessen and Marburg, have succeeded in identifying a new mode of action directed against coronaviruses (CoV): They were able to show that the substance thapsigargin efficiently inhibits the replication of highly pathogenic coronaviruses, including SARS-CoV-2.
While success in vaccine development is an essential building block in combating the corona pandemic, there are as yet no satisfactorily effective drugs that could rapidly lower viral load, limit the extent of COVID-19 disease, and thus reduce the risk of mortality. Working groups of the pharmacological and virological institutes of the Department 11 - Medicine of the Justus Liebig University Giessen (JLU) have now succeeded in identifying a new mode of action directed against coronaviruses (CoV). The scientists were able to show - in collaboration with a working group from the Philipps University of Marburg (UMR) and thanks to close networking in RNA virus research networks - that the substance thapsigargin efficiently inhibits the replication of highly pathogenic coronaviruses, including SARS-CoV-2. The researchers were recently able to publish the results of their study in the renowned journal Nature Communications.
The researchers now hope that their findings will contribute to the development of effective anti-CoV drugs. "The experimental work was consistently pursued even under pandemic conditions and makes an innovative contribution to research into antiviral agents against coronaviruses," pharmacologist and DZL scientist Prof. Dr. Michael Kracht, lead last author of the study, is convinced. The results demonstrated the high, internationally competitive level of coroanvirus research at the Central Hesse site, as well as the very good networking in the local RNA virus research networks supported by the Clinical Research Unit (KFO) 309 Virus-induced Lung Failure: Pathobiology and New Therapeutic Strategies, the Collaborative Research Center (SFB) 1021, the Pandemic Network Hesse, and the Von Behring-Röntgen Foundation, Marburg.
The starting point for the experiments were observations suggesting a close link between virus replication and changes in the endoplasmic reticulum (ER), one of the largest cell organelles. The ER has molecular sensing systems that can detect misfolded proteins, protein aggregates and foreign proteins. An adaptive ER stress response should allow it to compensate for cellular stress and help the cell return to its normal state. "When coronaviruses enter a cell, they initiate a massive remodeling of intracellular membranes to force the cells to form numerous so-called double-membrane vesicles (DMVs), in whose cavities the production of viral components then begins," explains Prof. Ziebuhr, also last author of this study, whose research group has long been internationally recognized in coronavirus research.
Antiviral effect discovered through thapsigargin
This rapid remodeling does not go unnoticed by the cell and evokes a massive activation of the ER stress system. "In turn, CoV reduce the protein levels of more than 150 proteins from the ER stress pathway within 24 hours," explains Prof. Kracht, whose research group studies intracellular signal transduction of cells in inflammation and infection models.
To find out whether this effect was virus-specific, the scientists used thapsigargin, a natural substance from the plant Thapsia garganica, since it was known that this substance also triggers a specific form of ER stress. In some experiments, thapsigargin was also added to the infected cells. Surprisingly, thapsigargin did not potentiate the virus-induced ER response, but exhibited a strong antiviral effect. "This rather incidental observation by molecular biologist and PhD student Mohammed Samer Shaban from our group, a first author of the publication, was the key experiment of our study," explains Prof. Kracht. Collaboration between the pharmacology and virology groups then demonstrated that thapsigargin effectively blocked replication not only of HCoV-229E, a less pathogenic cold virus, but also of the highly pathogenic MERS-CoV and SARS-CoV-2.
Thapsigargin more effective than remdesivir
"We placed particular emphasis on demonstrating these antiviral effects also in primary human cells from the bronchial epithelium of the respiratory tract, since the latter correspond to the natural site of infection of coronaviruses," adds virologist and further first author Dr. Christin Müller from the Ziebuhr group. Thapsigargin has long been studied for its potential use as an anticancer agent, as it is highly toxic to cells. "With this knowledge, extensive dose-response studies have been conducted in diverse cell systems, showing that the antiviral effects of thapsigargin occur at very low concentrations. Under thapsigargin, viral titers drop 100- to 1,000-fold. Even a single dose is sufficient to completely stop viral replication for up to three days. In addition, thapsigargin is ten times more effective than remdesivir against SARS-CoV-2," explains biologist Dr. Christin Mayr-Buro from AG Kracht, also first author.
Virus multiplication is inhibited by Thapsigargin
But how does thapsigargin work mechanistically? To this end, an investigation of the proteome of infected and thapsigargin-treated cells was carried out in collaboration with chemist Dr. Uwe Linne, who heads the mass spectrometry department of the Department of Chemistry at the University of Marburg and, together with Prof. Kracht, the central proteomics project of SFB1021. Using state-of-the-art techniques, more than 5,000 cellular proteins were measured from minute sample amounts of infected cells. Bioinformatic and functional analyses showed that thapsigargin improves the metabolism of the infected cells and additionally inhibits the process of autophagy.
"In principle, thapsigargin not only stops the rapid multiplication of viruses in the cell, but also reprograms this cell in such a way that it can survive a lethal viral infection for a longer period of time," Prof. Kracht states and classifies the scientific added value of the study: "Even if thapsigargin should not be clinically applicable in the end, our research results clearly show that anti-CoV therapeutics must block many switch points simultaneously in order to be effective. This chemical programming of an infected cell at many levels thus provides an important pharmacological mode of action for drugs targeting RNA viruses." The researchers involved are convinced that the joint results are thus equally relevant for basic and translational research.
Original publication: Multi-level inhibition of coronavirus replication by chemical ER stress